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HLA high-resolution gene sequencing kit

An HLA-A, HLA-DRB1 technology, applied in the field of specific primers, can solve the problems of high reagent cost, inability to distinguish, and inability to clearly combine, etc., to achieve fewer combinations of typing results, improve resolution and accuracy, and reduce The effect of reagent cost

Inactive Publication Date: 2010-11-24
SUZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The currently imported HLA-SBT reagents only sequence the HLA exons (Exon 2, 3). When the nucleotide differences of several alleles are outside these regions, it is impossible to distinguish them, resulting in inconsistencies in the experimental results. many undefined combinations (ambiguous results)
In addition, the reagents of SBT are all imported from abroad, and the cost of reagents is high

Method used

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  • HLA high-resolution gene sequencing kit
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  • HLA high-resolution gene sequencing kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Adopt PCR amplification primer and sequencing primer described in nucleotide sequence table (the position of described primer is as follows figure 1 , 2 shown), perform high-resolution genotyping of HLA-A, B, and DRB1 loci on human blood samples, and the specific steps are as follows:

[0102] (1) Genomic DNA extraction

[0103] Extraction was performed according to the operating instructions of Promaga's Genomic DNA Extraction Kit.

[0104] (2) PCR amplification

[0105]Genomic DNA extracted in step (1) was separately used with different primer pairs. Use HLA-A site amplification primers (SEQ ID No.1 and SEQ ID No.2) to carry out the amplification of HLA-A typing; Use HLA-B site amplification primers (SEQ ID No.3, SEQ ID No.3, SEQ ID No.2) No.4, SEQ ID No.5) to amplify HLA-B typing; use HLA-DRB1 locus amplification primers (SEQ ID No.6 to SEQ ID No.15) to amplify HLA-DRB1 typing Increase; above-mentioned amplification process and amplification reaction system are ...

Embodiment 2

[0128] High-resolution genotyping of HLA-A, B, and DRB1 loci was performed on the same blood sample using the HLA sequencing kit from Abbott, the United States, and the HLA sequencing kit and method in Example 1, respectively.

[0129] The sequencing maps of HLA-A (exons 2, 3, and 4), the sequencing maps of HLA-B (exons 2, 3, and 4), and the sequencing maps of exon 2 of HLA-DRB1 were obtained respectively.

[0130] The sequence diagram (test group) obtained by using the HLA sequencing kit and method in Example 1 is Figure 10 to Figure 16 The sequence diagram (control group) that adopts the HLA sequencing kit of Abbott Company of the United States to obtain is Figure 17 to Figure 23 .

[0131] Comparing the sequencing diagrams of exons 2, 3, and 4 of the HLA-A gene, we can see that:

[0132] Signal intensity of HLA-A Exon2 forward and reverse sequencing peaks in the control group: A2F A(232)G(227)C(279)T(345); A2R A(155)G(361)C(276)T(217 );

[0133] The signal intensity o...

Embodiment 3

[0151] Example 3, the experimental results of the international quality control of UCLA University in the United States and the quality control of the China Bone Marrow Bank

[0152] The HLA quality control samples of the China Bone Marrow Bank are basically allelic combinations common to Chinese people, and no missed detection was found with the reagent of the present invention, which is completely consistent with the result of the control reagent. The HLA quality control samples of UCLA University in the United States include the HLA allele combinations of various races around the world, especially the rare alleles in the Chinese population such as A*0260, B*1535, etc., and the results are also comparable to those of the control reagents. conform to.

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Abstract

The invention discloses a parting method of leucocyte antigen gene of human being, comprising the following steps of: (1) extracting genome DNA to be tested by a regular technology, and amplifying a destination gene fragment to be analyzed by using PCR amplification primer: 2,3,4 exon of HLA-A, 2,3,4 exon of HLA-B and exon on the locus 2 of HLA-DRB; and (2) amplifying the PCR output obtained in the step (1) by using sequencing primer, amplifying the exon, sequencing the amplified exon and comparing the sequencing result with the standard sequence in a database to determine the gene parting result. As the 2,3,4 exon of HLA-A, 2,3,4 exon of HLA-B and exon on the locus 2 of HLA-DRB are effectively amplified as a result of optimized combination of the HLA gene sequencing kit and the test condition, and the corresponding exon is sequenced, the invention solves the problem that effective parting can not be performed when certain allelic gene nucleotide is located outside an amplification area during further parting, thereby improving the parting resolution and accuracy of the HLA gene.

Description

[0001] technical field [0002] The invention belongs to the field of molecular biology, and relates to a method for amplifying and typing Human Leucocyte Antigen (Human Leucocyte Antigen, HLA)-A, B and DRB1 genes, and specific primers used in the method. Background technique [0003] HLA is the main gene system that regulates the body's specific immune response and determines individual differences in disease susceptibility. It plays an important role in antigen recognition, antigen presentation, immune response and regulation, and destroys foreign antigen target cells. It is the cause of immune rejection. The main material basis of the reaction. Both HLA-I and HLA-II antigens on the surface of graft cells are strong transplantation antigens. Both humoral immunity and cellular immunity are involved in the rejection of grafts. Whether it is allogeneic hematopoietic stem cell transplantation or organ transplantation, donor and recipient The degree of matching between HLA is ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 邱桥成
Owner SUZHOU UNIV
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